Abstract The hallmark of allergic diseases is the infiltration and accumulation of Th2 cells at the sites of inflammation; however the molecular mechanisms governing tolerance induction in Th2 cells and their relation to allergic responses are poorly understood. Thus, today it is becoming clear that understanding the Th2 cell biology and ways to control their function may prove to be invaluable for the treatment of allergic inflammation. Recently, we as well as others have demonstrated the role of E3 ubiquitin ligases such as Grail and Itch in induction of Th2 cell tolerance. Cullin4A (Cul4A) belongs to the evolutionally conserved E3 ubiquitin ligase family and has been suggested to be involved in hematopoietic cell activation and in chromatin regulation; however, the role of Cul4A in T lymphocytes has not been addressed yet. Interestingly, expression of Cul4A was upregulated in tolerant T cells. Moreover, Cul4A knockout CD4+ T cells activated in vitro under tolerogenic conditions exhibited an enhanced level of proliferation and IL-2 production compared to wild-type cells, suggesting an important role of Cul4A in regulation of T cell tolerance. Strikingly, we found that Cul4A deficient Th2 and Treg cells expressed higher levels of Th2 cytokines, which correlates with increased levels of IgG1 and IgE in the sera of Cul4A deficient mice, indicating the selective role of Cul4A in controlling Th2 programming and Th2 mediated inflammation. We also detected reduced Cul4A expression in CD4+ T cells from asthmatic patients compared to CD4+ T cells from healthy donors, further indicating that Cul4A potentially regulates the tolerogenic process in pro-allergic Th2 cells. In addition, shRNA knockdown of Cul4A in human CD4+ T cells resulted in upregulated expression of Th2-specific cytokines. Based on these findings, we hypothesize that regulation of Th2 cell function by Cul4A may be an important checkpoint in airway tolerance induction, which is crucial to prevent development of allergic diseases. In Aim 1, we propose to determine the molecular mechanisms whereby Cul4A controls T cell tolerance by utilizing gene knockdown approaches and in vitro and in vivo Th2 tolerance induction models. In Aim2, we will determine the role of Cul4A in Th2 cell programming and its underlying mechanisms as well. We will employ cutting edge technologies, including two-hybrid screening and microarray analysis of gene expression, to identify the exact target(s) of Cul4A to determine its function in Th2 development. In addition, we will assess the cellular mechanisms whereby Cul4A controls Th2 type mediated airway inflammation. In Aim3, we will determine the mechanism by which Cul4A regulates Tregs function, particularly the acquisition of Th2-like properties. The physiological significance of this finding will be analyzed in three different in vivo models. The proposed research will provide new significant insight into characterization of molecular mechanisms underlying Cul4A-mediated tolerogenic process in Th2 cells that may potentially have therapeutic implications for allergic diseases.